PROJECT SUMMARY Asthma is a highly prevalent chronic inflammatory airway disease that is genetically and mechanistically linked to allergy and dysregulated cytokine-mediated communication between the airway epithelial barrier and tissue-infiltrating immune cells. Th2 cells and their signature cytokines have long been implicated in the pathogenesis of asthma, and recent research indicates that Th17 cells may also be involved, at least in some disease endotypes. Improving our understanding of the molecular programming of Th2 and Th17-driven inflammation may lead to novel approaches for asthma therapy or prevention. MicroRNAs are tiny regulators of gene expression that mediate powerful biological effects through concerted action on networks of target mRNAs. We developed and deployed a robust functional screening platform for uncovering miRNA regulators of helper T cell differentiation and cytokine production. These screens yielded several miRNAs that we subsequently showed to be potent determinants of helper T cell responses, and several more that will be investigated in the proposed research. The central objective of this proposal is to leverage empirically determined target networks of these miRNAs to discover genes and pathways critical to the immunopathology of asthma. Guided by strong preliminary data, we will determine the physiological function(s) of several specific miRNA families (miR-24, miR-27, miR-18, miR-181, miR-130 and miR-30) in Th2 and Th17 differentiation, effector function, and ability to support pathology in mouse models of asthma. We will generate transcriptome-wide maps of miRNA binding using comparative Ago2 HITS-CLIP and test the cis-regulatory activity of those sites in high-throughput functional assays for 3? UTR function (FAST-UTR). These data will facilitate microRNA-directed discovery of genes and pathways involved in airway inflammation. GPR174, a G protein coupled lysophosphatidylserine receptor targeted by miR-24 and miR-27, will be the first novel miRNA target discovered in this way to be the subject of detailed analysis in vitro and in mice. Finally, we will use our newly developed platform for genome editing in primary human T cells to test the functional impact of individual miRNA:target interactions. We expect the proposed research to generate novel insights about helper T cell biology and asthma immunopathogenesis. In addition, our studies will advance our fundamental understanding of the network properties of miRNA regulation of gene expression and cell behavior.